Nickel-induced transcriptional memory in lung epithelial cells promotes interferon signaling upon nicotine exposure.

Exposure to nickel, an environmental respiratory toxicant, is associated with lung diseases including asthma, pulmonary fibrosis, bronchitis and cancers. Our previous studies have shown that a majority of the nickel-induced transcriptional changes are persistent and do not reverse even after the termination of exposure. This suggested transcriptional memory, wherein the cell 'remembers' past nickel exposure. Transcriptional memory, due to which the cells respond more robustly to a previously encountered stimulus has been identified in a number of organisms. Therefore, transcriptional memory has been described as an adaptive mechanism. However, transcriptional memory caused by environmental toxicant exposures has not been well investigated. Moreover, how the transcriptional memory caused by an environmental toxicant might influence the outcome of exposure to a second toxicant has not been explored. In this study, we investigated whether nickel-induced transcriptional memory influences the outcome of the cell's response to a second respiratory toxicant, nicotine. Nicotine, an addictive compound in tobacco, is associated with the development of chronic lung diseases including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Our results show that nicotine exposure upregulated a subset of genes only in the cells previously exposed to nickel. Furthermore, our analyses indicate robust activation of interferon (IFN) signaling in these cells. IFN signaling is a driver of inflammation, which is associated with many chronic lung diseases. Therefore, our results suggest that nicotine exposure of lung cells that retain the transcriptional memory of previous nickel exposure could result in increased susceptibility to developing chronic inflammatory lung diseases.

Interpretable predictive models of genome-wide aryl hydrocarbon receptor-DNA binding reveal tissue-specific binding determinants.

The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand-activated AhR binds to DNA at dioxin response elements (DREs) containing the core motif 5'-GCGTG-3'. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor (TF)-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible TFs.

MicroRNA-Gene Interactions Impacted by Toxic Metal(oid)s during EMT and Carcinogenesis.

Chronic environmental exposure to toxic metal(loid)s significantly contributes to human cancer development and progression. It is estimated that approximately 90% of cancer deaths are a result of metastasis of malignant cells, which is initiated by epithelial-mesenchymal transition (EMT) during early carcinogenesis. EMT is regulated by many families of genes and microRNAs (miRNAs) that control signaling pathways for cell survival, death, and/or differentiation. Recent mechanistic studies have shown that toxic metal(loid)s alter the expression of miRNAs responsible for regulating the expression of genes involved in EMT. Altered miRNA expressions have the potential to be biomarkers for predicting survival and responses to treatment in cancers. Significantly, miRNAs can be developed as therapeutic targets for cancer patients in the clinic. In this mini review, we summarize key findings from recent studies that highlight chemical-miRNA-gene interactions leading to the perturbation of EMT after exposure to toxic metal(loid)s including arsenic, cadmium, nickel, and chromium.

Nickel-induced alterations to chromatin structure and function.

Nickel (Ni), a heavy metal is prevalent in the atmosphere due to both natural and anthropogenic activities. Ni is a carcinogen implicated in the development of lung and nasal cancers in humans. Furthermore, Ni exposure is associated with a number of chronic lung diseases in humans including asthma, chronic bronchitis, emphysema, pulmonary fibrosis, pulmonary edema and chronic obstructive pulmonary disease (COPD). While Ni compounds are weak mutagens, a number of studies have demonstrated the potential of Ni to alter the epigenome, suggesting epigenomic dysregulation as an important underlying cause for its pathogenicity. In the eukaryotic nucleus, the DNA is organized in a three-dimensional (3D) space through assembly of higher order chromatin structures. Such an organization is critically important for transcription and other biological activities. Accumulating evidence suggests that by negatively affecting various cellular regulatory processes, Ni could potentially affect chromatin organization. In this review, we discuss the role of Ni in altering the chromatin architecture, which potentially plays a major role in Ni pathogenicity.

Oxidative stress modulates expression of immune checkpoint genes via activation of AhR signaling.

Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.

Transcriptional repression of E-cadherin in nickel-exposed lung epithelial cells mediated by loss of Sp1 binding at the promoter.

E-cadherin plays a central role in the stability of epithelial tissues by facilitating cell-cell adhesion. Loss of E-cadherin expression is a hallmark of epithelial-mesenchymal transition (EMT), a major event in the pathogenesis of several lung diseases. Our earlier studies showed that nickel, a ubiquitous environmental toxicant, induced EMT by persistently downregulating E-cadherin expression in human lung epithelial cells and that the EMT remained irreversible postexposure. However, the molecular basis of persistent E-cadherin downregulation by nickel exposure is not understood. Here, our studies show that the binding of transcription factor Sp1 to the promoter of E-cadherin encoding gene, CDH1, is essential for its expression. Nickel exposure caused a loss of Sp1 binding at the CDH1 promoter, resulting in its downregulation and EMT induction. Loss of Sp1 binding at the CDH1 promoter was associated with an increase in the binding of ZEB1 adjacent to the Sp1 binding site. ZEB1, an EMT master regulator persistently upregulated by nickel exposure, is a negative regulator of CDH1. CRISPR-Cas9-mediated knockout of ZEB1 restored Sp1 binding at the CDH1 promoter. Furthermore, ZEB1 knockout rescued E-cadherin expression and re-established the epithelial phenotype. Since EMT is associated with a number of nickel-exposure-associated chronic inflammatory lung diseases including asthma, fibrosis and cancer and metastasis, our findings provide new insights into the mechanisms associated with nickel pathogenesis.

Epithelial-mesenchymal transition: Insights into nickel-induced lung diseases.

Nickel compounds are environmental toxicants, prevalent in the atmosphere due to their widespread use in several industrial processes, extensive consumption of nickel containing products, as well as burning of fossil fuels. Exposure to nickel is associated with a multitude of chronic inflammatory lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. In addition, nickel exposure is implicated in the development of nasal and lung cancers. Interestingly, a common pathogenic mechanism underlying the development of diseases associated with nickel exposure is epithelial-mesenchymal transition (EMT). EMT is a process by which the epithelial cells lose their junctions and polarity and acquire mesenchymal traits, including increased ability to migrate and invade. EMT is a normal and essential physiological process involved in differentiation, development and wound healing. However, EMT also contributes to a number of pathological conditions, including fibrosis, cancer and metastasis. Growing evidence suggest that EMT induction could be an important outcome of nickel exposure. In this review, we discuss the role of EMT in nickel-induced lung diseases and the mechanisms associated with EMT induction by nickel exposure.

Nickel-induced transcriptional changes persist post exposure through epigenetic reprogramming.

BACKGROUND: Nickel is an occupational and environmental toxicant associated with a number of diseases in humans including pulmonary fibrosis, bronchitis and lung and nasal cancers. Our earlier studies showed that the nickel-exposure-induced genome-wide transcriptional changes, which persist even after the termination of exposure may underlie nickel pathogenesis. However, the mechanisms that drive nickel-induced persistent changes to the transcriptome remain elusive. RESULTS: To elucidate the mechanisms that underlie nickel-induced long-term transcriptional changes, in this study, we examined the transcriptome and the epigenome of human lung epithelial cells during nickel exposure and after the termination of exposure. We identified two categories of persistently differentially expressed genes: (i) the genes that were differentially expressed during nickel exposure; and (ii) the genes that were differentially expressed only after the termination of exposure. Interestingly, > 85% of the nickel-induced gene expression changes occurred only after the termination of exposure. We also found extensive genome-wide alterations to the activating histone modification, H3K4me3, after the termination of nickel exposure, which coincided with the post-exposure gene expression changes. In addition, we found significant post-exposure alterations to the repressive histone modification, H3K27me3. CONCLUSION: Our results suggest that while modest first wave of transcriptional changes occurred during nickel exposure, extensive transcriptional changes occurred during a second wave of transcription for which removal of nickel ions was essential. By uncovering a new category of transcriptional and epigenetic changes, which occur only after the termination of exposure, this study provides a novel understanding of the long-term deleterious consequences of nickel exposure on human health.

Role of CTCF in DNA damage response.

CCCTC-binding factor (CTCF) is a highly conserved, ubiquitously expressed zinc finger protein. CTCF is a multifunctional protein, associated with a number of vital cellular processes such as transcriptional activation, repression, insulation, imprinting and genome organization. Emerging evidence indicates that CTCF is also involved in DNA damage response. In this review, we focus on the newly identified role of CTCF in facilitating DNA double-strand break repair. Due to the large number of cellular processes in which CTCF is involved, factors that functionally affect CTCF could have serious implications on genomic stability. It is becoming increasingly clear that exposure to environmental toxicants could have adverse effects on CTCF functions. Here we discuss the various ways that environmental toxicants could impact CTCF functions and the potential consequences on DNA damage response.

Cadmium exposure upregulates SNAIL through miR-30 repression in human lung epithelial cells.

Cadmium (Cd) is a known human lung carcinogen. In addition, Cd exposure is associated with several lung diseases including emphysema, chronic obstructive pulmonary disease (COPD), asthma and fibrosis. Although earlier studies have identified several processes dysregulated by Cd exposure, the underlying mechanisms remain unclear. Here, we examined the transcriptome of lung epithelial cells exposed to Cd to understand the molecular basis of Cd-induced diseases. Computational analysis of the transcriptome predicted a significant number of Cd-upregulated genes to be targets of miR-30 family miRNAs. Experimental validation showed downregulation of all the miR-30 family members in Cd exposed cells. We found SNAIL, an EMT master regulator, to be the most upregulated among the miR-30 targets. Furthermore, we found decrease in the levels of epithelial marker E- cadherin (CDH1) and increase in the levels of mesenchymal markers, ZEB1 and vimentin. This suggested induction of EMT in Cd exposed cells. Luciferase reporter assays showed that miR-30 repressed SNAIL by directly targeting its 3' UTR. Over expression of miR-30e and transfection of miR-30e mimics reduced Cd-induced SNAIL upregulation. Our results suggest that miR-30 negatively regulates SNAIL in lung epithelial cells and that Cd-induced downregulation of miR-30 relieves this repression, resulting in SNAIL upregulation and EMT induction. EMT plays a major role in many diseases associated with Cd exposure including fibrosis, COPD, and cancer and metastasis. Therefore, our identification of miR-30 downregulation in Cd exposed cells and the consequent activation of SNAIL provides important mechanistic insights into lung diseases associated with Cd exposure.

Nickel exposure induces persistent mesenchymal phenotype in human lung epithelial cells through epigenetic activation of ZEB1.

Nickel (Ni) is an environmental and occupational carcinogen, and exposure to Ni is associated with lung and nasal cancers in humans. Furthermore, Ni exposure is implicated in several lung diseases including chronic inflammatory airway diseases, asthma, and fibrosis. However, the mutagenic potential of Ni is low and does not correlate with its potent toxicity and carcinogenicity. Therefore, mechanisms underlying Ni exposure-associated diseases remain poorly understood. Since the health risks of environmental exposures often continue post exposure, understanding the exposure effects that persist after the termination of exposure could provide mechanistic insights into diseases. By examining the persistent effects of Ni exposure, we report that Ni induces epithelial-mesenchymal transition (EMT) and that the mesenchymal phenotype remains irreversible even after the termination of exposure. Ni-induced EMT was dependent on the irreversible upregulation of ZEB1, an EMT master regulator, via resolution of its promoter bivalency. ZEB1, upon activation, downregulated its repressors as well as the cell-cell adhesion molecule, E-cadherin, resulting in the cells undergoing EMT and switching to persistent mesenchymal status. ZEB1 depletion in cells exposed to Ni attenuated Ni-induced EMT. Moreover, Ni exposure did not induce EMT in ZEB1-depleted cells. Activation of EMT, during which the epithelial cells lose cell-cell adhesion and become migratory and invasive, plays a major role in asthma, fibrosis, and cancer and metastasis, lung diseases associated with Ni exposure. Therefore, our finding of irreversible epigenetic activation of ZEB1 by Ni exposure and the acquisition of persistent mesenchymal phenotype would have important implications in understanding Ni-induced diseases.

Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells.

Mercury (Hg) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are major environmental contaminants that commonly co-occur in the environment. Both Hg and TCDD are associated with a number of human diseases including cancers. While the individual toxicological effects of Hg and TCDD have been extensively investigated, studies on co-exposure are limited to a few genes and pathways. Therefore, a significant knowledge gap exists in the understanding of the deleterious effects of co-exposure to Hg and TCDD. Due to the prevalence of Hg and TCDD co-contamination in the environment and the major human health hazards they pose, it is important to obtain a fuller understanding of genome-wide effects of Hg and TCDD co-exposure. In this study, by performing a comprehensive transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) exposed to Hg and TCDD individually and in combination, we have uncovered a subset of genes with altered expression only in the co-exposed cells. We also identified the additive as well as antagonistic effects of Hg and TCDD on gene expression. Moreover, we found that co-exposure impacted several biological and disease processes not affected by Hg or TCDD individually. Our studies show that the consequences of Hg and TCDD co-exposure on the transcriptional program and biological processes could be substantially different from single exposures, thus providing new insights into the co-exposure-specific pathogenic processes.

Rapid Recall Ability of Memory T cells is Encoded in their Epigenome.

Even though T-cell receptor (TCR) stimulation together with co-stimulation is sufficient for the activation of both naïve and memory T cells, the memory cells are capable of producing lineage specific cytokines much more rapidly than the naïve cells. The mechanisms behind this rapid recall response of the memory cells are still not completely understood. Here, we performed epigenetic profiling of human resting naïve, central and effector memory T cells using ChIP-Seq and found that unlike the naïve cells, the regulatory elements of the cytokine genes in the memory T cells are marked by activating histone modifications even in the resting state. Therefore, the ability to induce expression of rapid recall genes upon activation is associated with the deposition of positive histone modifications during memory T cell differentiation. We propose a model of T cell memory, in which immunological memory state is encoded epigenetically, through poising and transcriptional memory.

Oxidative stress under ambient and physiological oxygen tension in tissue culture.

Oxygen (O2) levels range from 2-9% in vivo. However, cell culture experiments are performed at atmospheric O2 levels (21%). Oxidative stress due to generation of reactive oxygen species (ROS) in cells cultured at higher than physiological levels is implicated in multitude of deleterious effects including DNA damage, genomic instability and senescence. In addition, oxidative stress activates redox sensitive transcription factors related to inflammatory signaling and apoptotic signaling. Furthermore, several chromatin-modifying enzymes are affected by ROS, potentially impacting epigenetic regulation of gene expression. While primary cells are cultured at lower O2 levels due to their inability to grow at higher O2, the immortalized cells, which display no such apparent growth difficulties, are typically cultured at 21% O2. This review will provide an overview of issues associated with increased oxygen levels in in vitro cell culture and point out the benefits of using lower levels of oxygen tension even for immortalized cells.

Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung Epithelial Cells at Atmospheric Oxygen Levels.

Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels.

Lineage relationship of CD8(+) T cell subsets is revealed by progressive changes in the epigenetic landscape.

To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8(+) T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8(+) T-cell subsets. Taken together, our results suggest that CD8(+) lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory.

Epigenetic dysregulation by nickel through repressive chromatin domain disruption.

Investigations into the genomic landscape of histone modifications in heterochromatic regions have revealed histone H3 lysine 9 dimethylation (H3K9me2) to be important for differentiation and maintaining cell identity. H3K9me2 is associated with gene silencing and is organized into large repressive domains that exist in close proximity to active genes, indicating the importance of maintenance of proper domain structure. Here we show that nickel, a nonmutagenic environmental carcinogen, disrupted H3K9me2 domains, resulting in the spreading of H3K9me2 into active regions, which was associated with gene silencing. We found weak CCCTC-binding factor (CTCF)-binding sites and reduced CTCF binding at the Ni-disrupted H3K9me2 domain boundaries, suggesting a loss of CTCF-mediated insulation function as a potential reason for domain disruption and spreading. We furthermore show that euchromatin islands, local regions of active chromatin within large H3K9me2 domains, can protect genes from H3K9me2-spreading-associated gene silencing. These results have major implications in understanding H3K9me2 dynamics and the consequences of chromatin domain disruption during pathogenesis.

Insulators recruit histone methyltransferase dMes4 to regulate chromatin of flanking genes.

Chromosomal domains in Drosophila are marked by the insulator-binding proteins (IBPs) dCTCF/Beaf32 and cofactors that participate in regulating long-range interactions. Chromosomal borders are further enriched in specific histone modifications, yet the role of histone modifiers and nucleosome dynamics in this context remains largely unknown. Here, we show that IBP depletion impairs nucleosome dynamics specifically at the promoters and coding sequence of genes flanked by IBP binding sites. Biochemical purification identifies the H3K36 histone methyltransferase NSD/dMes-4 as a novel IBP cofactor, which specifically co-regulates the chromatin accessibility of hundreds of genes flanked by dCTCF/Beaf32. NSD/dMes-4 presets chromatin before the recruitment of transcriptional activators including DREF that triggers Set2/Hypb-dependent H3K36 trimethylation, nucleosome positioning, and RNA splicing. Our results unveil a model for how IBPs regulate nucleosome dynamics and gene expression through NSD/dMes-4, which may regulate H3K27me3 spreading. Our data uncover how IBPs dynamically regulate chromatin organization depending on distinct cofactors.

Chromatin immunoprecipitation indirect peaks highlight long-range interactions of insulator proteins and Pol II pausing.

Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks" of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.

A novel human polycomb binding site acts as a functional polycomb response element in Drosophila.

Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.